Abstract Fam20C is the archetypical member of a unique family of secretory pathway kinases and is critical for many cellular processes including proper biomineralization of bones and teeth and regulation of kidney phosphate metabolism. Inactivating mutations in Fam20C cause Raine Syndrome, a severe osteosclerotic bone dysplasia that presents as abnormal hardening of bones, microcephaly, and gum hyperplasia. Fam20C phosphorylates secretory/extracellular proteins within a highly conserved Ser-x-Glu/pSer motif. We have recently discovered that Fam20C kinase activity is allosterically regulated via formation of a protein complex with a closely related member of the Fam20 family, Fam20A. Inactivating Fam20A mutations also cause severe tooth enamel defects in the disorder Amelogenesis Imperfecta, suggesting that both Fam20C and Fam20A are critical for proper biomineralization. We have queried the secreted phosphoproteome from several cell types and found that Fam20C is responsible for generating the vast majority (>90%) of secretory phosphoproteins. The secreted phosphoproteome is highly cell-type specific and the biological functions of Fam20C phosphorylation within most of these proteins are unclear. Many of the Fam20C substrates that we have identified are components of the extracellular matrix, and loss of Fam20C function exerts profound effects on cellular adhesion, migration, and invasiveness. Together, our preliminary data suggest that Fam20C- dependent phosphorylation plays critical roles in processes extending far beyond biomineralization to other diverse functions such as regulation of the extracellular matrix, wound healing, and lipid homeostasis. Because little is known about the role of specific Fam20C phosphorylation events within target substrates, our findings highlight the importance of understanding how Fam20C is regulated and of clarifying the physiological impact of Fam20C phosphorylation of specific substrates. This proposal will focus on defining the regulation of Fam20C by Fam20A and determining the role of Fam20C phosphorylation sites within extracellular matrix proteins.